Norovirus (NoV), also known previously as “Norwalk-Like Virus” (NLV) or small round structured virus, is the most important viral pathogen of epidemic acute gastroenteritis that occurs in both developed and developing countries. These genetically diverse viruses comprise two major genogroups (GI and GII) and approximately 30 genotypes. NoVs belong to the genus Norovirus in the Caliciviridae family and are non-enveloped, icosahedral, single stranded, positive-sense RNA viruses whose outer protein capsids are composed of 180 copies of a single major structural protein, the VP1 protein.
The NoV genome is ˜7.6 kb in length, and is composed of three open reading frames (ORFs), of which the latter two encode the major (VP1) and minor (VP2) structural proteins of NoV capsid, respectively. When expressed in vitro in eukaryotic cells, the NoV capsid protein self-assembles into virus-like particle (VLPs) that are structurally and antigenically indistinguishable from native NoV virions. The crystal structure of NoV capsid reveals a T=3 icosahedral symmetry, formed by 180 VP1s that organize into 90 dimers (17). Each VP1 can be divided into two major domains, the shell (S) and the protruding (P) domains that constitute the icosahedral shell and protruding arches of the capsid, respectively. The protruding arch, formed by a P dimer, represents the surface antigenic structure of NoV and is responsible for virus-host interactions and immune responses of NoVs. NoV VLPs serve as a useful tool for NoV research, owing to the lack of an effective cell culture and a small animal model for human NoVs. The S and P domains appear to be structurally and functionally independent. Expression of the S domain alone forms S particles with a smooth surface without binding function to histo-blood group antigens (HBGAs), the viral receptors or ligands of human NoVs. On the other hand, expression of the P domain (with or without end-modifications) can form three types of P domain complexes, each having binding function to HBGAs. They are the P dimer, and two larger oligomers of the P dimer: the 12-mer small P particle, and the 24-mer P particle. We have also recently identified 18-mer and 36-mer P complexes, demonstrating the interchangeable nature and dynamic relationship of all P domain complexes. Since the P dimer and the P particle can be easily produced in Escherichia coli (E. coli) and retain HBGA-binding function, they have been used as models for the study of NoV-HBGA interaction extensively. U.S. Pat. No. 8,277,819, the disclosure of which is incorporated by reference in its entirety, describes Norovirus capsid protein monomers having only the P domain that can assemble spontaneously into a P-particle having an icosahedral form. These stable P-particles are useful in methods for diagnosing and treating Norovirus-infected individuals, and in methods for making vaccines and for the treatment, amelioration and prevention of Norovirus infections.
Previously it has been demonstrated that the P particle can be applied as a vaccine platform for foreign antigen presentation. Each P domain has three surface loops on the distal end, corresponding to the outermost surface of the P particle. Previous studies demonstrated that these loops are excellent sites in presenting a foreign antigen for increased immune responses. U.S. Pat. Publication 2010-0322962, which is incorporated herein by reference in its entirety, discloses that a distal portion of the NoV P-domain monomer includes a peptide string into which a peptide unit of a foreign antigen, and in particular a foreign viral antigen, can be inserted. The resulting antigen-P-domain monomers can spontaneously assemble into a nanoparticle called an antigen-P-particle, typically of an octahedral form, that consists of 24 of the antigen-P-domain monomers arranged into 12 dimers. This P-particle is easily produced in E. coli, extremely stable, and highly immunogenic. We studied this particle using a His-tag as a model and obtained excellent results.
Bioengineering has become an important field that advances many technologies of modern medicine. Development of recombinant viral subunit vaccines for control and prevention of infectious diseases is a typical example. Unlike traditional vaccines, which are either live attenuated or inactivated viruses, the subunit vaccines are recombinant viral proteins. Therefore, the subunit vaccines do not have risk of infection while inducing protective antiviral immune responses and thus represent a new, safer generation of vaccines. Successful examples of such recombinant vaccines include the four commercially available virus-like particle (VLP) vaccines: Recombivax HB (Merck) and Energix-B (GlaxoSmithKline, GSK) against hepatitis B virus (HBV) and Gardasil (Merck) and Cervarix (GSK) against human papilloma virus (HPV). Additionally, numerous other subviral vaccines, including the Norovirus (NoV) VLP and P particle vaccines are under intensive development. Hence, recombinant subunit vaccine technology represents an innovative vaccine strategy complementary to conventional vaccine approaches.
An important factor for a recombinant viral antigen to become an effective vaccine is its immunogenicity. Most icosahedral VLPs are highly immunogenic because of their large sizes and polyvalent antigenic structures. However, many other monomeric, dimeric and oligomeric viral antigens possess a low immunogenicity due to their smaller sizes and low valences. Traditionally, these small antigens need to be presented by a large, multivalent vaccine platform for improved immunogenicity to become candidate vaccines. For example, rotavirus VP8* antigen (159 residues), the outermost portion of the spike protein VP4, has been conjugated to the surface loop of the 24-meric NoV P particle for increased immunogenicity and protective immunity. However, although a number of small viral or bacterial antigens have been successfully presented by different multivalent platforms, limitations clearly exist depending on the structural compatibility between the antigens and the platforms, which prevent a wider application of the current vaccine platforms.
Notwithstanding the advancements in the therapeutic treatment of and vaccine development against viral and bacterial infection, including NoV infections, there remains a need for improving and/or enhancing the immunogenicity of vaccines in general, and in particular providing a vaccine against NoV that has increased immunogenicity. It would also be beneficial to provide a large, multivalent immunogenic composition that can significantly increase the functionality of a functional group, such as a drug effective group, and/or enhance the immunogenicity of an antigen or epitope. There also remains a need to provide therapeutic treatment in the form of a multivalent vaccine for infections caused by NoV, as well as virus types other than NoV.